33 USE OF ADULT MESENCHYMAL STEM CELLS FOR CLONING PIGS

Abstract

Current cloning efficiencies are relatively low and there is evidence to suggest that a less differentiated cell type can increase these (Jaenisch et al. 2002 Cloning and Stem Cells 4, 389-396). As part of an ongoing study that aims to develop cloning as a breeding tool for the pig industry, we examined whether mesenchymal stem cells (MSCs) isolated from bone marrow (Experiment 1) and blood (Experiment 2) could increase cloning efficiencies compared with fibroblasts isolated from ear tissue of live animals. MSCs were isolated from the femurs and blood of two pigs over a Histopaque-1077 density gradient. Cells were seeded and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics/antimycotics. Blood MSCs were plated onto fibronectin-coated dishes (Faast and Nottle, Proc. Australian Health Med. Res. Congress 2004, abst. 1272). Cells from passages 2-4 were used for nuclear transfer by means of a fusion before being subjected to the activation protocol described previously (Harrison et al. 2004 Cloning and Stem Cells 6, 327-331). Embryos were cultured in a modified NCSU23 for 7 days. In Experiment 1 there was no difference in the number of fused embryos that cleaved in the bone marrow MSC and fibroblast groups (374/447; 84% vs. 370/446; 83%, respectively; 9 replicates). The number of embryos that developed to the blastocyst stage by day 7 was significantly higher in the bone marrow MSC group compared with the fibroblast group (136/447; 30% vs. 79/446; 18%, respectively, Chi square test; P < 0.01). In Experiment 2 there was no difference in the percentage of embryos that cleaved in the blood MSC and fibroblast groups (287/375; 77% vs. 275/347; 79%, respectively; 8 replicates). The number of embryos that developed to the blastocyst stage by day 7 was also similar between the blood MSC and fibroblast groups (67/375; 18% vs. 63/347; 18%, respectively). In conclusion, our results suggest that bone marrow MSCs may be a more efficient cell type, compared with fibroblasts, for the cloning of live animals.